We have shown that dual beam flow cytometry may be a powerful tool for classification of chromosomes isolated from human cells. The aims of this project are to optimize procedures for dual beam flow cytometric classification of human chromosomes (bivariate flow karyotyping) and to begin evaluation of the clinical and research uses of this technique. Optimization efforts will include: 1) Design and construction of a simple-to-operate, inexpensive dual beam flow cytometer for analysis of human chromosomes stained with Hoechst 33258 and chromomycin A3 or dyes with similar spectral properties. The new cytometer will be 5.7 times more efficient in fluorescence collection than our current system and will require only a single laser; 2) Improvement of procedures for human lymphocyte culture and for chromosome isolation; 3) Optimization of existing chromosome staining procedures to permit maximum discrimination among human chromosomes. In these studies, Hoechst 33258 will be compared to Hoechst 33342 and DAPI and chromomycin A3 will be compared to mithramycin and olivomycin. We will also study the addition of methyl green or distamycin A to our current dual staining protocol as a way of discriminating between normally occurring polymorphisms and structural rearrangements; 4) Development of a fast, robust computer analysis procedure for estimation of the bivariate mean and volume in a bivariate flow karyotype, even if the peak is superimposed on a debris continuum. Bivariate flow karyotyping will be evaluated by analyzing a battery of fibroblasts and lymphocytes containing a variety of clinically important karyotypic rearrangements including: 1) Structural rearrangements such as Robertsonian translocations, reciprocal translocations, deletions and insertions. In addition, flow karyotypes will be measured for several individuals with retinoblastoma (deletion in chromosome 13) or Wilm's tumor (deletion in chromosome 11); and 2) Numerical aberrations such as trisomy 21, 18, 13 and sex chromosome aneuploidy. These analyses, together with analyses of normal cells and cells of unknown karyotype (blind study) should allow preliminary evaluation of the utility of bivariate flow karyotypes in the detection and study of clinically important karyotypic abnormalities.